WO2005012533A2 - Genotoxicity testing - Google Patents
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- WO2005012533A2 WO2005012533A2 PCT/GB2004/003337 GB2004003337W WO2005012533A2 WO 2005012533 A2 WO2005012533 A2 WO 2005012533A2 GB 2004003337 W GB2004003337 W GB 2004003337W WO 2005012533 A2 WO2005012533 A2 WO 2005012533A2
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
Definitions
- the present invention relates to improved methods for detecting agents that cause or potentiate DNA damage and to molecules and transformed cells that may be usefully employed in such methods.
- DNA damage is induced by a variety of agents such as ultraviolet light, X rays, free radicals, methylating agents and other mutagenic compounds. These agents may cause damage to the DNA that comprises the genetic code of an organism and cause mutations in genes. In microorganisms such mutations may lead to the evolution of new undesirable strains of the microorganism. For instance, antibiotic or herbicide resistant bacteria may arise. In animals these mutations can lead to carcmogenesis or may damage the gametes to give rise to congenital defects in offspring.
- agents such as ultraviolet light, X rays, free radicals, methylating agents and other mutagenic compounds. These agents may cause damage to the DNA that comprises the genetic code of an organism and cause mutations in genes. In microorganisms such mutations may lead to the evolution of new undesirable strains of the microorganism. For instance, antibiotic or herbicide resistant bacteria may arise. In animals these mutations can lead to carcmogenesis or may damage the gametes to give rise to congenital defects in offspring.
- DNA damaging agents may chemically modify the nucleotides that comprise DNA and may also break the phosphodiester bonds that link the nucleotides or disrupt association between bases (T-A or C-G).
- T-A or C-G The DNA damaging agents may chemically modify the nucleotides that comprise DNA and may also break the phosphodiester bonds that link the nucleotides or disrupt association between bases (T-A or C-G).
- a method of detecting these agents may be used as a mutagenesis assay for screening compounds that are candidate medicaments, food additives or cosmetics to assess whether or not the compound of interest induces DNA damage.
- methods of detecting DNA damaging agents may be used to monitor for contamination of water supplies with pollutants that contain mutagenic compounds.
- WO 98/44149 An improved genotoxicity test is disclosed in WO 98/44149.
- This specification concerns recombinant DNA molecules comprising a regulatory element that activates gene expression in response to DNA damage operatively linked to a DNA sequence that encodes a light emitting reporter protein.
- DNA molecules may be used to transform a cell and such cells used in a genotoxic test for detecting for the presence of an agent that causes or potentiates DNA damage.
- the cells may be subjected to an agent and the expression of the light emitting reporter protein from the cell indicates that the agents cause DNA damage.
- the genotoxicity tests described in WO 98/44149 detect the induction of DNA repair activity, which evolved to minimise conversion of damage to mutation.
- the method described in WO 98/44149 may therefore be used to detect for the presence of DNA damaging agents.
- WO 98/44149 describes a number of useful genetic constructs that may be used to tranform a cell such that it may be used in a genotoxic test.
- One such construct is yEGFP-444 (illustrated in Figure 12 of WO 98/44149), and the present invention is based upon a novel type of construct that was identified during developmental work carried out with yEGFP-444.
- a recombinant vector comprising a recombinant DNA molecule comprising a DNA sequence that encodes a light emitting reporter protein, which DNA sequence is operatively linked to a regulatory element arranged to activate expression of the DNA sequence in response to DNA damage, wherein when used to transform a cell, the vector does not substantially alter the sensitivity of the cell to geneticin, when compared to the sensitivity of the cell which has not been transformed with the vector.
- a method of generating a recombinant vector comprising the steps of:- (i) providing a vector backbone with a DNA sequence that encodes a light emitting reporter protein; (ii) operatively linking the DNA sequence to a regulatory element arranged to activate expression of the DNA sequence in response to DNA damage; (iii) providing the vector backbone with a selectable marker arranged to confer resistance to geneticin; and (iv) rendering the selectable marker non-functional, wherein when used to transform a cell, the vector does not substantially alter the sensitivity of the cell to geneticin, when compared to the sensitivity of the cell which has not been transformed with the vector.
- a method of detecting for the presence of an agent that causes or potentiates DNA damage comprising subjecting a cell in accordance with the third aspect of the present invention to an agent and monitoring the expression of the light emitting reporter protein from the cell.
- regulatory element we mean a DNA sequence that regulates the transcription of a gene with which it is associated.
- operatively linked we mean that the regulatory element is able to induce the expression of the reporter protein.
- reporter protein we mean a protein which when expressed in response to the regulatory element of the DNA molecule of the invention is detectable by means of a suitable assay procedure.
- pWDH445 we mean the expression vector illustrated in Figure 5 of this specification.
- pWDH445 was originally called yEGFP444 and was originally disclosed in Figure 12 of WO 97/44149.
- the sensitivity to geneticin of a cell which has been transformed with the vector according to the first aspect of the invention is at least 70% that of the sensitivity to geneticin of a cell which has not been transformed with the vector.
- the sensitivity to geneticin is at least 80%, more preferably, at least 90%, even more preferably at least 95%, and most preferably, at least 99% that of the sensitivity to geneticin of a cell which has not been transformed with the vector.
- the sensitivity to geneticin of a cell which has been transformed with the vector according to the first aspect is the same as that of the sensitivity to geneticin of a cell which has not been transformed with the vector.
- the resistance to geneticin of a cell transformed with the vector is the same as that of the resistance to geneticin of the cell which has not been transformed with the vector.
- the method of the fourth aspect of the invention represents a novel cost- effective genotoxicity screen, that may be used to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens.
- the method of the fourth aspect of the invention is suitable for assessing whether or not an agent may cause DNA damage. It is particularly useful for detecting agents that cause DNA damage when assessing whether it is safe to expose a person to DNA damaging agents. For instance, the method may be used as a mutagenesis assay for screening whether or not known agents, such as candidate medicaments, foodstuffs or cosmetics, induce DNA damage. Alternatively, the method of the fourth aspect of the invention may be used to monitor for contamination of water supplies with pollutants containing DNA damaging agents.
- the method of the fourth aspect of the invention may equally be used for assessing whether an agent may potentiate DNA damage.
- certain agents can cause accumulation of DNA damage by inhibiting DNA repair (for instance by preventing expression or function of a repair protein) without directly inflicting DNA damage. These agents are often known as co-mutagens.
- the present invention is based upon work conducted on the vectors disclosed in WO 98/44149.
- the inventors have found that a spontaneous rearrangement of the known vector pWDH445 occurred that resulted in a surprisingly brighter reporter.
- reporter we mean the degree of fluorescence from the reporter is surprisingly at least double that of the known vector pWDH445, more preferably at least 3 times, at least 5 times, at least 7 times, at least 10 times, and most preferably, at least 12 times that of the known vector pWDH445.
- the inventors therefore conducted experiments, which are described in more detail in the Examples, to characterise this fortuitous mutation and to isolate recombinant vectors according to the first aspect of the present invention.
- the vector pWDH445 is illustrated in Figure 5.
- the inventors have found that, surprisingly, loss of the kanMXi module function (i.e. removing geneticin resistance) from the pWDH445 plasmid results in a vector capable of expressing the reporter protein (GFP in the case of pWDH445) such that the signal from the reporter is significantly greater when DNA damage occurs.
- the kanMX3 module may be disrupted such that a mutation occurs by means of deletion, substitution or addition of nucleotides provided that geneticin resistance is impaired.
- the vector may comprise nucleotide bases of the kanMX3 module, but the gene is disrupted such that it is non-functional.
- the vector according to the first aspect preferably comprises a light emitting reporter protein, a regulatory element and a non-functional kanMX module.
- Discovery of the loss of kanMX3 module function in vector pWDH445 led the inventors to realise that vectors according to the first aspect of the invention were particularly useful for genotoxicity testing. They also devised the method according to the second aspect for generating prefe ⁇ ed vectors according to the first aspect, which vectors may be used in a method according to the fourth aspect.
- the vector backbone used in the method of the second aspect may comprise any suitable vector backbone known to those skilled in the art, which may be used to cany the light emitting reporter protein, the regulatory element, and the selectable marker conferring resistance to geneticin.
- the backbone may comprise a low copy number or high copy number plasmid.
- the backbone may be selected from the preferably well-known vectors pWDH445, pWDH443 (Walmsley et al, R.M., 1997), pRS316 (Sikorski et al., 1989).
- Recombinant vectors according to the first aspect may be designed such that they may autonomously replicate in the nucleus of the cell.
- elements, which induce DNA replication may be required in the recombinant vector.
- the vector may comprise an origin of replication, preferably, for yeast. Suitable origins of replication will be known to the skilled technician.
- a suitable element is derived from the yeast 2 ⁇ plasmid.
- Such replicating vectors can give rise to multiple copies of the DNA molecule in a transformant cell and are therefore useful when over-expression (and thereby increased light emission) of the reporter protein is required.
- the transformant cell will be the cell according to the third aspect.
- the recombinant vector may comprise at least one selectable marker to enable selection of cells transfected with the vector, and preferably, to enable selection of cells harbouring the recombinant vector that incorporates the DNA molecule of the first aspect.
- selectable markers include genes conferring resistance to an antibiotic, for example, kanamycin, and ampicillin etc.
- selectable markers may include auxotrophic markers, i.e. those which restore prototrophy, for example, yeast URA3 or LETJ2 genes.
- the selectable marker conferring geneticin resistance may comprise any of the kanMX modules, for example, kanMX2, kanMX3, kanMX4, kanMX ⁇ .
- prefe ⁇ ed vectors according to the first aspect of the invention comprise pWDH445 (y EGFP-444 in WO 98/44149) with disruption of the kanMX3 module.
- the DNA sequence that encodes a light emitting reporter protein may code for any light emitting protein. However, it is prefe ⁇ ed that the DNA sequence codes for a protein that is fluorescent. Prefe ⁇ ed DNA sequences that encode a light emitting reporter protein code for Green Fluorescent Protein (GFP) and light emitting derivatives thereof. GFP is from the jellyfish Aequorea victoria and is able to absorb blue light and re-emits an easily detectable green light and is thus suitable as a reporter protein. GFP may be advantageously used as a reporter protein because its measurement is simple and reagent free and the protein is non-toxic.
- GFP Green Fluorescent Protein
- Derivatives of GFP include DNA sequences encoding for polypeptide analogues or polypeptide fragments of GFP, which are able to emit light. Many of these derivatives absorb and re-emit light at wavelengths different to GFP found endogenously in Aequorea victoria.
- preferred DNA molecules according to the first aspect of the invention have a DNA sequence that encodes the S65T derivative of GFP (in which serine 65 of GFP is replaced by a threonine).
- S65T GFP has the advantage that it is brighter than wild-type GFP (when excited at its longest- wavelength peak) and shows only slow photobleacbing.
- S65T GFP produces a good quantum yield of fluorescence and matches the output of argon ion lasers used in fluorescence activated cell sorters.
- Cells according to the second aspect of the invention which contain DNA molecules coding S65T GFP may be used according to the method of the fourth aspect of the invention and are particularly useful when light emission is measured from cell extracts.
- a most prefe ⁇ ed DNA sequence encodes for a yeast enhanced GFP (yEGFP) such as the GFP derivative described by Cormack et al. (1997) (in Microbiology 143 p303-311).
- yEGFP has an amino acid sequence, which is biased for usage in yeast.
- yEGFP is particularly suitable for transforming cells according to the second aspect of the invention which are yeast.
- light emitted from yEGFP in such yeast is even greater than that emitted by S65T derivatives.
- Recombinant DNA molecules coding yEGFP are also useful because yEGFP is less heat sensitive than nascent GFP.
- the regulatory element of the recombinant DNA molecule activates expression of the reporter protein when DNA damage occurs.
- Such regulatory elements ideally comprise a promoter sequence, which induces RNA polymerase to bind to the DNA molecule and start transcribing the DNA encoding the reporter protein.
- the regulatory element may also comprise other functional DNA sequences such as translation initiation sequences for ribosome binding or DNA sequences that bind transcription factors which promote gene expression following DNA damage. Regulatory elements may even code for proteins, which act to dislodge inhibitors of transcription from the regulated gene and thereby increase transcription of that gene.
- Preferred regulatory elements are DNA sequences that are associated in nature with the regulation of the expression of DNA repair proteins.
- the regulatory elements from genes such as RAD2, RAD6, RAD7, RAD18, RAD23, RAD51, RAD54, CDC7, CDC8, CDC9, MAGI, PHRl, DINl, DDR48 and UB14 from yeast may be used to make recombinant DNA molecules according to the first aspect of the invention.
- the regulatory element used in the method of the second aspect may comprise genes such as RAD2, RAD6, RAD7, RAD 18, RAD23, RAD51, RAD54, CDC7, CDC8, CDC9, MAGI, PHRl, DINl, DDR48 or TJBI4 from yeast.
- a prefe ⁇ ed regulatory element comprises the promoter and 5' regulatory sequences of the RAD54 repair gene.
- a regulatory element may be derived from yeast and particularly Saccharomyces cerevisiae. It is most prefe ⁇ ed that the regulatory element comprises the promoter and 5' regulatory sequences of the RAD54 repair gene which correspond to the DNA sequence identified in WO 98/44149 or a functional analogue or fragment thereof.
- a preferred recombinant vector according to the first aspect comprises the RAD54 gene operatively linked to GFP, or light emitting derivative thereof. It is especially prefe ⁇ ed that the vector additionally comprises a non-functional kanMX3 module.
- most prefe ⁇ ed recombinant vector according to the first aspect of the invention is pGenOOl, as illustrated in Figure 15. The nucleotide sequence of pGenOOl is given in Figures 16 and 17.
- the recombinant DNA molecule in pGenOOl is interchangeable. Accordingly, the RAD54 promoter - yEGFP expression cassette depicted in Figure 15 may be easily replaced (e.g. by Bam HI / Ascl digestion) to insert another recombinant DNA molecule according to the invention (see below).
- the RAD54 regulatory element in pGenOOl may be replaced by an RNR regulatory element to provide a recombinant vector according to the first aspect.
- the resultant recombinant vector produces a surprisingly brighter signal of the light emitting reporter protein, when exposed to DNA damage.
- the regulatory element of the recombinant vector according to the first aspect and as used in the method of the second aspect, may comprise an RNR regulatory element.
- RNR regulatory element we mean a DNA sequence that is the natural regulator of an RNR gene.
- Prefe ⁇ ed vectors according to the invention comprise pWDH445 (yEGFP-444 in WO 98/44149) with disruption of the kanMX3 module in winch an RNR regulatory element replaces the RAD54 regulatory element.
- the kanMXi module may be disrupted such that a mutation occurs by means of deletion, substitution or addition of nucleotides provided that geneticin resistance is impaired.
- the RNR regulatory element of the recombinant DNA molecule activates expression of the reporter protein when DNA damage occurs.
- Such regulatory elements ideally comprise a promoter sequence, which induces RNA polymerase to bind to the DNA molecule and start transcribing the DNA encoding for the reporter protein.
- the regulatory element may also comprise other functional DNA sequences such as translation initiation sequences for ribosome binding or DNA sequences that bind transcription factors which promote gene expression following DNA damage. Regulatory elements may even code for proteins which act to dislodge inhibitors of transcription from the regulated gene and thereby increase transcription of that gene.
- Preferred regulatory elements are DNA sequences that are associated in nature with the regulation of the expression of RNR DNA repair proteins.
- the regulatory elements from genes such as RNR1, RNR2 and RNR3 from yeast may be used in the method of the second aspect to make recombinant DNA molecules according to the first aspect of the invention.
- the regulatory element may comprise an RNR1, RNR2 or RNR3 gene from yeast.
- a prefe ⁇ ed regulatory element comprises the promoter and 5' regulatory sequences of the RNR2 gene.
- the RNR2 gene may be found on chromosome X of Saccharomyces cerevisiae.
- a preferred regulatory element may be derived from between co-ordinates 387100 and 398299 associated with the RNR2 gene on chromosome X as identified in the Saccharomyces cerevisiae genome database. It is more preferably derived from between co-ordinates 387100 and 393299.
- the database may be accessed by the World Wide Web at many sites. For example at genome- www. Stanford, edu.
- the regulatory element of RNR3 is particularly prefe ⁇ ed.
- the sequence of this RNR3 element is well known and is illustrated in Figure 33.
- the gene is YIL066C, and the Figure 33 also shows lOOObp upstream of the ATG start codon, which is highlighted in bold.
- the total sequence length of RNR3 is 2610bp long.
- the RNR3 promoter is particularly suitable because its induction is DNA damage specific; there is low level expression under normal condition; and significant induction incurs in response to damage.
- a preferred recombinant vector according to the first aspect comprises the RNR2 or RNR3 gene operatively linked to GFP, or light emitting derivative thereof. It is especially prefe ⁇ ed that the vector additionally comprises a non-functional kanMX3 module.
- a most preferred recombinant vector according to the first aspect of the invention is pGenRNR2, as illustrated in Figure 24.
- the nucleotide sequence of pGenRNR2 is given in Figure 26.
- Another prefe ⁇ ed recombinant vector according to the first aspect of the invention is pGenRNR3, as illustrated in Figure 25.
- the nucleotide sequence of pGenRNR3 is given in Figure 27.
- most prefe ⁇ ed recombinant vectors comprise RAD54, RNR2 or RNR3 regulatory element operatively linked to a DNA sequence encoding GFP or a light emitting derivative thereof, which preferably further comprises a non-functional kanMX3 module.
- the recombinant vector according to the first aspect of the present invention may for example be a plasmid, cosmid or phage.
- Such recombinant vectors are of great utility when replicating the DNA molecule.
- recombinant vectors are highly useful for transforming cells with the DNA molecule and may also promote expression of the reporter protein.
- each of the vectors is able to autonomously replicate in a host yeast cell due to the presence of a 2 ⁇ element. It will be appreciated that, due to legislation involving use of genetically modified organisms, it is especially prefe ⁇ ed that the vectors are only used in an enclosed environment, and not released in to the environment.
- Figure 34 shows pGenOOl ( Figure 34a) being digested with Apa LI to produce the vector pGenEMOOl, and is shown in Figure 34b.
- a similar digestion of pGenRNR2 or pGenRNR3 with Apa LI will also remove the bacterial origin of replication and ampicillin resistance gene.
- the recombinant vector may be designed such that the vector and DNA molecule of the first aspect integrate into a chromosome of the host cell.
- Such integration has the advantage of improved stability compared to replicative plasmids.
- DNA sequences, which favour targeted integration are desirable.
- incorporation into the recombinant vector of fragments of the HO gene from chromosome IV of S.cerevisiae favours targeted integration in S. cerevisiae or cell lines derived therefrom. It is prefe ⁇ ed that the fragment of the HO gene has the sequence as shown in Figure 35, or a derivative thereof.
- a prefe ⁇ ed vector may therefore comprise the RAD54 gene or the RNR2 gene or the RNR3 gene, operatively linked to GFP, or a light emitting derivative thereof, a non-functional kanMX3 module, and a nucleotide sequence adapted to integrate into the genome of a target cell.
- the nucleotide sequence may be a fragment of the HO gene.
- the LEU2-d gene (the Saccaromyces cerevisiae wild type LEJJ2 gene with a 29bp truncated promoter) was amplified with flanking Aatll cloning sites from the plasmid pEMBL-yex4 (Cesareni et al, 1987). This fragment was cloned into pWDH443 (Wahnsley et al, 1997) to give the plasmid pGenlnOll. This plasmid was further modified by removing 2.1kb of the KanMX module by Sac I digestion and religation, resulting in a non-functional KanMX module. This plasmid was named pGenh 012.
- pGenfr ⁇ 012 a prefe ⁇ ed integrating vector according to the invention is referred to as pGenfr ⁇ 012, and is illustrated in Figure 36. The full sequence of this vector is shown in Fig:41, and is 7515 bp in length.
- pGenfr ⁇ 012 comprises RAD54 and it will be appreciated that similar integrating vectors comprising RNR2 or RNR3 although not illustrated are in accordance with the invention.
- pGenfr ⁇ 012 comprises a LEU2-d selectable marker and a non-functional kanMX3 module.
- DNA sequences which favour targeted integration into the genome, and which may be incorporated into the recombinant vector include sequences from the ribosomal DNA array of S.cerevisiae. Such rDNA sequences favour targeted integration in to chromosome XII of S.cerevisiae, or cell lines derived therefrom. It is prefe ⁇ ed that the rDNA sequence has the sequence as shown in Figure 37, or is a derivative thereof.
- a 4.5kb Bgl II cut rDNA fragment from the Saccaromyces cerevisiae genome was cloned into Bam HI cut pGenh ⁇ 012 refe ⁇ ed to above. As this fragment was cloned in two different orientations (which lead to different orientations of integration) the two different forms of these plasmids were named pGenIn022A and pGenh ⁇ 022B.
- the plasmid, pGenfr ⁇ 022A was cut with Sph I prior to transformation into the yeast strain FF 18984 to facilitate plasmid integration within the tandem rDNA repeats of the Saccaromyces cerevisiae genome.
- a prefe ⁇ ed vector may therefore comprise the RAD54 gene or the RNR2 gene or the RNR3 -gene, operatively linked to GFP, or a light emitting derivative thereof, a non-functional kanMX3 module, and a nucleotide sequence adapted to integrate into the genome of a target cell, wherein the nucleotide sequence may be rDNA sequence.
- pGenh ⁇ 022 A-form a prefe ⁇ ed integrating vector according to the invention is referred to as pGenh ⁇ 022 A-form, and is illustrated in Figure 38.
- the full sequence of this vector is shown in Fig:42, and is 12093 bp in length.
- pGenfr ⁇ 022 A-form comprises RAD54 and it will be appreciated that similar integrating vectors comprising RNR2 or RNR3 although not illustrated are in accordance with the invention.
- pGen_h022A comprises the LEU2-d selectable marker and a non-functional kanMX3 module.
- Figure 40 illustrates (i) the effect of the presence of kanMX on the brightness of pGenh ⁇ 012, i.e.
- rDNA B int and rDNA A int - A and B refer to orientation of the reporter cassette in the chromosome. It is evident that rDNA A int - A is the brightest construct, possibly due to effects of the surrounding DNA sequence and protein interactions therewith, such as steric hindrance.
- recombinant vectors may be formed from pFA vectors or derivatives thereof, which are known to the art (see Wach et al. (1994) Yeast 10 pl793-1808).
- Preferred recombinant vectors are derived from yEGFP-444 disclosed in WO 97/44149 (the disclosure of which is incorporated herein by reference). yEGFP-444 is also known as pWD ⁇ 445.
- the recombinant vector is incorporated within a cell.
- host cells may be prokaryotic or eukaryotic. Suitable host cells include bacteria, plant, yeasts, insect and mammalian cells. Prefe ⁇ ed host cells are yeast cells such as Saccharomyces cerevisiae. Yeast are prefe ⁇ ed because they can be easily manipulated like bacteria but are eukaryotic and therefore have DNA repair systems that are more closely related to humans than those of bacteria. Another benefit of using yeast cells as a host is that DNA repair systems are inducible in yeast unlike in humans where the repair systems are largely constitutive.
- Preferred yeast cells include: (i) Y485 in haploid form; (ii) Y486 (also l ⁇ iown as FF18984) in haploid form; (iii) Y485/486 in diploid form; (iv) FY73 (v) YLR030w.al ⁇ ha.; and (vi) Y300. These strains may all be found in national yeast strain collections.
- a preferred strain comprises GenTOl.
- the reporter strain comprises the yeast FF18984 containing a replicative plasmid (pGenOOl shown in Figure 15) containing the entire upstream non-coding DNA sequence of the RAD54 gene fused to the yeast-enhanced Aequorea victoria GFP gene.
- Host cells used for expression of the protein encoded by the DNA molecule are ideally stably transformed, although the use of unstably transformed (transient) cells is not precluded.
- Transformed cells according to the third aspect of the invention may be fonned by following procedures described in the Example.
- the cell is ideally a yeast cell (for instance one of the strains described above).
- Such transformed cells may be used according to the method of the fourth aspect of the invention to assess whether or not agents induce or potentiate DNA damage.
- GFP expression is induced in response to DNA damage and the light emitted by GFP may be easily measured using a fluorimeter as an index of the DNA damage caused. For instance, the light emitted by GFP at 511 nm (after excitation between 475 and 495 nm— e.g.
- 488 nm in response to DNA damage, may be evaluated either in a suspension of a defined number of whole cells or from a defined amount of material released from cells following breakage.
- light emitted by GFP at 520 nm may be evaluated through a 535nm filter.
- the method of the fourth aspect of the invention is particularly useful for detecting agents that induce DNA damage at low concentrations.
- the methods may be used to screen compounds, such as candidate medicaments, food additives or cosmetics, to assess whether it is safe to expose a living organism, particularly people, to such compounds.
- the method of the fourth aspect of the invention may be employed to detect whether or not water supplies are contaminated by DNA damaging agents or agents that potentiate DNA damage.
- the methods may be used to monitor industrial effluents for the presence of pollutants that may lead to increased DNA damage in people or other organisms exposed to the pollution.
- the cells according to the second aspect of the invention are ideally unicellular organisms such as bacteria, algae, protozoa and particularly yeast.
- the expression of light emitting reporter protein may be monitored according to the method of the invention from cell extracts or from samples containing intact, whole cells.
- the method of the invention is preferably performed by growing cells transformed with a recombinant vector according to the first aspect of the invention (such as pGenOOl, pGenRNR2, ⁇ GenRNR3, pGenEMOOl, pGenh ⁇ 012, or pGenfr ⁇ 022A), incubating the cells with the agent which putatively causes DNA damage for a predetermined time and monitoring the expression of the light emitting reporter protein directly from a sample of the cells.
- a recombinant vector according to the first aspect of the invention such as pGenOOl, pGenRNR2, ⁇ GenRNR3, pGenEMOOl, pGenh ⁇ 012, or pGenfr ⁇ 022A
- prefe ⁇ ed yeast according to the third aspect of the invention may be grown in FI medium (described in Walmsley et al. (1983) Mol. Gen. Genet. 192 p361-365 and the Example).
- FF18984 cells may be transformed with pGenOOl and grown in FI medium.
- FF 18984 cells may be transformed with pGenRNR2 or ⁇ GenRNR3 and grown in FI medium.
- FF18984 may be transformed with any of pGenEMOOl, pGenh ⁇ 012, or pGenIn022A, and grown in FI medium.
- a putative DNA damaging agent e.g. a food additive or potential medicament or an agent contained within a water sample or effluent sample
- the cells are then allowed to grow for a defined period of time after which a sample of the cells is removed and fluorescence measured therefrom. This measurement may be effected by estimating the cell concentration and fluorescence in the sample using nephelometry (light scattering). For example, cells can be illuminated at 600 nm and the scattered light (at 600 nm) estimated at 90 degrees to the incident beam. The light emitted by GFP can be measured by excitation at 475-495 mn (e.g.
- the method of the invention should ideally employ sensitive fluorimeters and reduce light scattering in order that light emission can be accurately measured from the reporter protein.
- sensitivity can be improved by using a 487 nm filter which is introduced between the sample chamber and the emission-detector of the fluorimeter. Such a filter further reduces the impact of light scattering and improves the sensitivity of the method when samples containing whole cells are used.
- a preferred method of testing for DNA damage comprises the steps of : (1) preparing a microplate for use in an assay; (2) conducting the assay in the microplates; (3) collecting and analysing the data; and (4) making a judgment on DNA damage and the consequences.
- Microplate preparation Assays were carried out in 96 well, black, clear-bottomed microplates. For example Matrix ScreenMates, Cat. No. 4929, Apogent Discoveries, USA, or Corning (BV, Netherlands: Cat. No. 3651). A number of alternative microplates were assessed, though the variable background absorbance and fluorescence both within and between plates from individual manufacturers were generally unacceptable, leading to the cu ⁇ ently prefe ⁇ ed choice. It will therefore be appreciated that microplates used according to the invention preferably have consistent absorbance and fluorescence between plates and batches thereof.
- the assay plates can be filled using a liquid handling robot.
- a liquid handling robot For example the MicroLabS single probe, from Hamilton GB Ltd., Birmingham or a Genesis 8-probe robot (Tecan UK Ltd. Theale. UK).
- Microplates can also be filled rapidly and effectively using a multi-channel pipette.
- the cytotoxicity threshold is set at 80 % of the cell density reached by the untreated control cells. This is greater than 3 times the standard deviation of the background.
- a positive cytotoxicity result (+) is concluded if 1 or 2 compound dilutions produce a final cell density lower than the 80% threshold.
- a strong positive cytotoxicity result positive (++) is concluded when either (i) three or more compound dilutions produce a final cell density lower than the 80% threshold or (ii) at least one compound dilution produces a final cell density lower than a 50% threshold.
- a negative result (-) is concluded when no compound dilutions produce a final cell density lower than the 80% threshold.
- the lowest effective concentration (LEC) is the lowest test compound concentration that produces a final cell density below the 80% threshold.
- the compound absorbance control allows a warning to be generated if a test compound is significantly absorbing. If the ratio of the absorbance of the compound control well to a well filled with diluent alone is > 2, there is a risk of interference with interpretation.
- the cytotoxicity controls indicate that the yeast is behaving normally.
- the 'high' methanol standard should reduce the final cell density to below the 80% threshold, and should be a lower value than the 'low' standard.
- the genotoxic threshold is set at a relative GFP induction of 1.3 (i.e. a 30% increase). Tins is greater than 3 times the standard deviation of the background.
- a positive genotoxicity result (+) is concluded if 1 or 2 compound dilutions produce a relative GFP induction greater than the 1.3 threshold.
- a strong positive genotoxicity result (++) is concluded if either (i) three or more compound dilutions produce a relative GFP induction greater than the 1.3 threshold or (ii) at least one compound dilution produces a relative GFP induction greater than a 1.6 threshold.
- a negative genotoxicity result (-) is concluded where no compound dilutions produce a relative GFP induction greater than the 1.3 threshold.
- the LEC is the lowest test compound concentration that produces a relative GFP induction greater than the 1.3 threshold.
- the genotoxic controls demonstrate that the strains are responding normally to DNA damage.
- the 'high' MMS standard must produce a fluorescence induction > 2, and be a greater value than the 'low' MMS standard.
- Anomalous brightness data is generated when the toxicity leads to a final cell density less than 30% that of the blank. Genotoxicity data is not calculated above this toxicity threshold. Compounds that tested negative for genotoxicity, were re-tested up to lOmM, or to the limit of solubility or cytotoxicity.
- the compound fluorescence control allows a warning to be generated when a compound is highly auto-fluorescent. If the ratio of the fluorescence of the compound control well to a diluent filled well is >5, there is a risk of interference with interpretation. In these cases (four in this study), fluorescence polarisation can be used to distinguish GFP from other sources of fluorescence (Knight et al, 2000, 2002). Both the Tecan and BMG instruments have this facility. Occasionally, compounds though not fluorescent themselves, induce cellular auto-fluorescence. This is apparent from rising brightness in the control (GenCOl) strain in the absence of fluorescence from the chemical-only control. The routine subtraction of GenCOl from GenTOl data removes this interference from the data.
- a recombinant vector comprising a recombinant DNA molecule comprising a regulatory element that activates gene expression in response to DNA damage operatively linked to a DNA sequence that encodes a light emitting reporter protein and a DNA vector characterised in that the vector comprises an origin of replication; at least one selectable marker; and when used to transform a cell, does not alter the sensitivity of the cell to geneticin.
- a recombinant vector comprising a recombinant DNA molecule comprising an RNR regulatory element operatively linked to a DNA sequence that encodes a light emitting reporter protein and a DNA vector characterised in that the vector comprises an origin of replication; at least one selectable marker; and when used to transfoim a cell, does not alter the sensitivity of the cell to geneticin.
- Figure 1 shows the difference between the brightness values obtained from FF18984 cells transformed with pWDH445, exhibiting normal levels of brightness ( ⁇ ) and enhanced brightness ( M ) without and with exposure to MMS.
- U untransformed FF18984 cells not treated with MMS;
- U MMS untransformed cells exposed to 0.005% MMS (15 hours);
- T cells transformed with pWDH445 but not treated with MMS;
- T MMS cells transformed with pWDH445 and exposed to 0.005% MMS;
- Brightness fluorescence intensity (F int) corrected for culture density using the intensity of scattered light at 600 nm (Neph600).
- Each bar represents the average brightness value of three independent cultures. Both uninduced and MMS -induced brightness values are clearly significantly enhanced in the new transformants relative to the original transformants.
- Figure 2 shows an ethidium bromide stained agarose gel of pWDH445 cut with Bam ⁇ . I and Ase I.
- the lanes contain the following: Lane 1, pWDH445; Lane 2, plasmid isolated from an enhanced brightness transformant; Lane 3, pWDH445 isolated from a normal brightness yeast transformant; and Lane 4, a second isolate from a transformant exhibiting enhanced brightness.
- BamR l-Asc I restriction of pWDH445 liberates a fragment of 3.2 kb (boxed) bearing HO-i iD5 ⁇ promoter- yEGFP cassette. A band representing a 3.2 kb-sized fragment is visible in all four lanes.
- Figure 3 shows FF18984 cells transformed with pWD ⁇ 445 and cells re-transformed with re-isolated pWDH445 or rearranged plasmid (two independent isolates) isolated from transformants of enhanced brightness.
- the four sets of transformants were replica-plated onto SD medium deficient in uracil and YPD medium incorporating 200 ⁇ gml "1 geneticin (G418) and incubated at 30°C for 3 days. All four sets of transformants grew on SD-ura but only the cells bearing pWDH445 and re-isolated pWDH445 grew on YPD+G418.
- Figure 4 demonstrates that FF18984 cells re-transformed with rearranged plasmid ( ⁇ ) are brighter than FF 18984 cells bearing pWDH445 ( ⁇ ) without and with exposure to 0.005% MMS.
- U untransformed FF 18984 cells not exposed to MMS;
- U MMS untransformed cells exposed to 0.005% MMS (15 hours);
- T cells transformed with pWDH445 or rea ⁇ anged plasmid not exposed to MMS;
- T MMS cells transformed with pWDH445 or rearranged plasmid and exposed to 0.005% MMS;
- Brightness fluorescence intensity (F int) corrected for culture density using the intensity of scattered light at 600 nm (Neph ⁇ OQ).
- Each bar represents the average brightness value of six independent cultures. Cells re-transformed with the re-isolated rearranged plasmid are still brighter than normal pWDH445 transformants both without and with exposure to MMS.
- Figure 5 shows the restriction map of pWDH445 created for and used in this study.
- Base-pair sequences of the plasmid constituents were analysed for restriction enzyme cleavage sites using software available on the Stanford Genome Database (SGD) web-site.
- Major plasmid components are shown as blocked sections with the plasmid backbone (mainly bacterial sequences, such as the origin of replication) drawn represented by a line (-).
- the RAD54 promoter ( ⁇ ) -yEGFP ( ⁇ ) cassette and the kanMX3 cassette ( B ) are shaded to highlight the main regions of interest, whilst Amp ⁇ and URA3 are represented by open block arrows.
- the a ⁇ ows represent the direction of transcription for individual components.
- Figure 6 shows an ethidium bromide stained agarose gel of the four plasmids (pWDH445, re-isolated pWDH445, and two rearranged plasmids) digested with_ ⁇ b ⁇ I.
- the lanes were loaded as follows: Lane 1, pWDH445; Lane 2, rearranged plasmid; Lane 3, re-isolated pWDH445; Lane 4, second rea ⁇ anged plasmid; Lane 5, 500 bp ladder (5-0.5 kb) marker DNA.
- Xba I restriction of both rearranged plasmids produced a novel band representing a fragment of ⁇ 4.4 kb, which was not detected for pWDH445 and re-isolated pWDH445.
- the rearranged plasmids also appear to be missing one band of a 6 kb doublet observed for the pWDH445 digestions.
- Extra bands in lane 2 reveal that one rearranged plasmid is larger than pWDH445, while the loss of a band from the doublet (and no extra bands) suggest that the rearranged plasmid loaded in lane 4 is smaller than pWDH445.
- Figure 7 shows an ethidium bromide stained agarose gel of the four plasmids (pWDH445, re-isolated pWDH445, and two rea ⁇ anged plasmids) restriction digested with Sea I.
- the lanes were loaded as follows: Lane 1, 500 bp ladder (5-0.5 kb) molecular weight marker DNA; Lane 2, pWDH445; Lane 3, larger rearranged plasmid; Lane 4, re-isolated pWDH445; Lane 5, smaller rearranged plasmid. Bands representing fragments sized -1.8 kb and -4.1 kb are present in all four lanes (lanes 2- 5) but the band representing a fragment of 1.3 kb (present for pWDH445 and re- isolated pWDH445) is not exhibited in the digestion of either rea ⁇ anged plasmid (lanes 3 and 5).
- Figure 8 presents an ethidium bromide stained agarose gel of the four plasmids (pWDH445, re-isolated pWDH445, and two rea ⁇ anged plasmids) restriction digested with Pst I. Lanes were loaded as follows: Lane 1, 500 bp ladder molecular weight marker DNA; Lane 2, pWDH445; Lane 3, larger rearranged plasmid; Lane 4, re- isolated pWDH445; Lane 5, smaller rea ⁇ anged plasmid. Lanes 2 and 4 produced identical banding patterns whilst those in lanes 3 and 5 (rearranged plasmids) are significantly different. Only lanes 2 and 4 exhibit a band representing a fragment of ⁇ 2.5 kb. Lanes 2 - 5 all exhibit bands representing fragments of -1.6, -1.4, and -1.3 kb, but lanes 3 and 5 also showed an extra band representing a fragment of 1.5 kb.
- Figure 9 shows an ethidium bromide stained agarose gel of Pvu I-digested pWDH445, re-isolated pWDH445, and rearranged plasmids. Lanes loaded as follows: Lane 1, pWDH445; Lane 2, larger rearranged plasmid; Lane 3, re-isolated pWDH445; Lane 4, smaller rea ⁇ anged plasmid; Lane 5, 500 bp ladder molecular weight marker DNA. Lanes 1 and 3 exhibited the expected banding pattern, whereas neither rearranged (lanes 2 and 4) plasmid exhibits bands representing fragments of 1.2 and 0.7 kb. The larger rea ⁇ anged plasmid (lane 2) also shows two novel bands representing fragments of -3.5 kb and 1 kb.
- Figure 10 shows an ethidium bromide stained agarose gel of the four plasmids (pWDH445, re-isolated pWDH445, and two rea ⁇ anged plasmids) digested with Sac I. Lanes were loaded with the following digested plasmids: Lane 1, pWDH445; Lane 2, larger rearranged plasmid; Lane 3, re-isolated pWDH445; Lane 4, smaller rea ⁇ anged plasmid; Lane 5, 500 bp ladder molecular weight marker DNA. Sac I liberates a 2.1 kb kanMX internal fragment from pWDH445 and both lanes 1 and 3 exhibit a band representing a 2.1 kb fragment. Neither rea ⁇ anged plasmid (lanes 2 and 4) reveals such a band after Sac I restriction.
- Figure 11 presents an ethidium bromide stained agarose gel of Xba I-digested pWDH445 and pWDH445 with kanMX enzymically removed. Lanes were loaded as follows: Lanes 1 and 13, pWDH445; Lanes 2-6, 8, 9, 11, and 12, plasmid isolated from ampicillin-resistant, kanamycin-sensitive E. coli transformants; Lane 10, plasmid isolated from an ampicillin-resistant, kanamycin-resistant E.coli transformant; Lane 7, 500 bp ladder molecular weight marker DNA. Lanes 1 and 13 exhibit a doublet band representing two fragments of -6 kb, which is also seen for the plasmid from the kanamycin-resistant colony (lane 10).
- Figure 12 represents the brightness values of FF18984 cells transformed with modified pWDH445 (enzymically removed kanMX) ( ⁇ ) compared with those from cells transformed with pWDH445 ( ⁇ ).
- Figure 13 shows a schematic of the flow-through fluorescence detector with a blown- up schematic of the detector orientations within the light-tight box, and a further blown-up cartoon of the excitation beam and emissions, relative to the flow cell.
- the top section shows the overall layout of the instrument, beginning with cultures incubating in a water-bath shaker. Alternatively, samples can be injected via the sample injection valve. Culture/sample is pumped peristaltically into the light-tight instrument enclosure (grey box), wherein excitation occurs and emission is detected.
- the source of the excitation beam is an argon-ion laser ( ⁇ ).
- ⁇ argon-ion laser
- a beam-splitting cube send excitation light (488 nm) to both sets of detectors, each of which consists of an optical flow cell, photomultiplier tube (PMT), silicon photodiode (SPD), and optical filters (see key panel for colour-coding). Fluorescence is detected by the PMT whilst scattered light (as a measure of cell or particulate density) is detected by the SPD, both of which are situated perpendicular to the direction of the excitation beam. Orientation of the excitation beam and the emission detection is simplified in the bottom panel.
- Figure 14 depicts the detection of fluorescence polarisation with the flow-through fluorescence detector.
- Sample circulating through the flow cell is excited by plane- polarised laser light (vertical arrows represent the plane of polarisation) and scattered light is detected by an SPD placed at the other side of the flow cell in line with the beam of excitation.
- Emitted fluorescence is less polarised than the excitation light and this is represented by vertical, horizontal, and diagonal arrows around the emission, before reaching the polaroid filters.
- the two polaroid filters either side of the flow cell are positioned in opposite orientations, such that one filter allows transmission of the vertical component of the fluorescence (Parallel orientation with respect to the plane polarised excitation light), whilst the other permits transmission of the horizontal component (Perpendicular orientation with respect to the plane of polarisation of the absorbed light). Fluorescence transmitted by the polaroid filter is detected by two PMTs (PMT1 and PMT2).
- Figure 15 shows the restriction map of pGenOOl a preferred recombinant vector according to the presentinvention.
- Figure 16 shows full sequence of pGenOOl in FASTA format
- Figure 17 shows full sequence of pGenOOl in GeneBank format
- Figure 18 represents data from Example 2 illustrating that Methyl methanesulfonate is strongly cytotoxic and genotoxic.
- Figure 19 represents data from Example 2 illustrating that Benzaldehyde is cytotoxic and genotoxic.
- Figure 20 provides Genotoxicity and cytotoxicity data from the Assay described in Example 2 including test concentration and limits of detection, with comparative data from other genotoxicity tests according to Example 2.
- Figure 21 provides extracted genotoxicity data comparing GreenScreen Assay and reported Ames Test results highlighting compounds requiring S9 metabolic activation for a positive Ames result according to Example 2.
- Figure 22 illustrates the Greenrack loading sequence according to Example 3.
- Figure 23 illustrates a microplate layout according to Example 3.
- Figure 24 shows the restriction map of pGenRNR2 a preferred recombinant vector according to the present invention.
- Figure 25 shows the restriction map of pGenRNR3 a preferred recombinant vector according to the present invention.
- Figure 26 shows the full sequence of pGenRNR2 in GeneBank format.
- Figure 27 shows the full sequence of pGenRNR3in GeneBank format.
- Figure 28 shows results for (A) a cuvette assay and; (B) a microplate assay for a test strain transfected with pGenRNR2 and using MMS as a test compound in Example 2.
- Figure 29 shows results for (A) a cuvette assay and; (B) a microplate assay for a test strain transfected with pGenRNR3 and using MMS as a test compound in Example 2.
- Figure 30 shows results for a microplate assay for a test strain transfected with pGenRNR3 and using MMS as a test compound in Example 4.
- Figure 31 shows results for a microplate assay for a test strain transfected with pGenRNR3 and using 1,2-Dimethylhydrazine as a test compound in Example 4.
- Figure 32 shows results for a microplate assay for a test strain transfected with pGenRNR3 and using Ethyl methanesulfonate as a test compound in Example 4.
- Figure 33 shows the full sequence of RNR3 sequence including lkb upstream of start codon in GeneBank format.
- Figure 34 shows the removal of bacterial origin of replication and Amp resistance from pGenOOl to generate pGenEMOOl, a prefe ⁇ ed vector according to the invention.
- Figure 35 shows a fragment of HO sequence.
- Figure 36 shows the restriction map of pGen_n012, a preferred recombinant vector according to the present invention.
- Figure 37 shows rDNA sequence used in multiple copy rDNA integrating plasmids according to the invention.
- Figure 38 shows the restriction map of pGen_h022A, a preferred recombinant vector according to the present invention.
- Figure 39 shows the difference between the brightness values obtained from empty pRS316 vector compared with pRS316 containing the RAD54-GFP reporter cassette plus the kanMX module, and compared with pRS316 containing the RAD54-GFP cassette but not kanMX.
- Figure 40 show the effect of the presence of kanMX on the brightness of RAD54- GFP (all without MMS) when integrated into the chromosome at HO ( ⁇ int) and the rDNA array (rDNA B int and rDNA A int - A and B refer to orientation).
- Figure 41 shows the full sequence of pGenIn012 in GeneBank format.
- Figure 42 shows the full sequence of pGenIn022A in GeneBank format.
- Plasmids pWDH445 - is illustrated in Figure 5 and corresponds to yEGFP-444 as disclosed in WO 97/44149 (e.g. see Figure 12)
- Yeast media Bacto-agar was added to a final concentration (w/v) of 2% to each of the growth media.
- Yeast extract, peptone, and agar were all obtained from Difco (Becton Dickinson, Sparks, MD 21152, USA). The following media were used (Sherman et al. "Methods in yeast genetics” Cold Spring Harbor Laboratory Press).
- YPD yeast extract, peptone, and dextrose Table 2 Component % w/v Final concentration gL " Bacto-yeast extract 1 10 Bacto-peptone 2 20 Dextrose (glucose) 2 20 *Bacto-agar 2 20)
- Bacto-yeast mfrogen base was purchased without amino acids or ammonium sulphate. This allows control of the nitrogen source and facilitates the preparation of selective media.
- a 20x YNB stock comprising 34gL _1 Bacto-yeast nitrogen base and 100 gL "1 (NH 4 ) 2 SO 4 was stored in the dark at 4°C.
- FI medium is a defined minimal medium used in fermentation [Brown et al, 1981, adapted by Walmsley et al, 1983] that has a particularly low background autofiuorescence.
- Concentrated stocks of salts, trace elements and iron (III) chloride were autoclaved, stored separately, and diluted into sterile water upon requirement.
- a concentrated vitamin stock was prepared in sterile water, filter sterilised by syringe and sterile 0.20 ⁇ m pore filter (Sartorius, Gottingen, Germany), and stored in aliquots at -20°C. Vitamins were then added subsequent to autoclaving of FI, to avoid their denaturation. All FI was stored at 4°C in the absence of glucose.
- a subsequent variant form of FI was employed with 50% less inositol than quoted in the vitamins stock Table 8 in order to lessen the effects of flocculation in troublesome yeast strains.
- This reduced inositol FI was further modified by producing it in phosphate buffer at pH6 (as described in Varley, 1967. Practical Clinical Biochemistry, 4th Edition, William Heinemann Medical Books, pp. 759), instead of in water.
- the buffered medium was created by mixing 0.067 M solutions of KH 2 PO 4 and Na 2 HPO 4 , or concentrated stocks thereof (see Table 9), before addition of salts, trace elements, iron chloride, and nutritional supplements.
- the buffer mix was autoclaved and allowed to cool down to room temperature before addition of vitamins.
- Luria-Bertani medium (LB) for broth cultures was prepared as described [Sambrook et al, second edition, 1989] and LB broth plus 2% Bacto-agar was used for solid cultures.
- Ampicillin was prepared in aqueous solution at a stock concentration of 10 mgml "1 and used at working concentrations of up to 100 ⁇ gml "1 . Ampicillin stock solutions were stored in aliquots of 1 ml at -20°C.
- G418 disulphate salt was prepared in aqueous solution as a stock solution at the concentration of 20 mgml "1 and used at a working concentration of 200 ⁇ g/ml.
- the G418 stock solution was stored at 4°C.
- Kanamycin was also prepared in aqueous solution, at a stock concenfration of 50 mgml "1 and used at a working concentration of 50 ⁇ gmi "1 . Kanamycin stock solutions were stored at -20°C.
- Standard yeast and E.coli techniques for example, biomass production, transformation, isolation of plasmid DNA, and restriction enzyme digests, were used in accordance with published methods.
- the cultures were adjusted to 0.02% NaN 3 to inhibit respiration and incubated on ice for 90 minutes, maintaining agitation.
- the cells were then transfe ⁇ ed to 1.5 ml microfuge tubes, harvested by centrifugation (10s), washed twice in sterile distilled water, then washed in 1 ml of "extraction" buffer (20 mM Tris-Cl pH 7.5, 0.1 M NaCl, and 1 mM EDTA). After harvesting the cells by centrifugation, the supernatant was aspirated off and the cell pellet resuspended in 250 ⁇ l "crushing" buffer (20 mM Tris-HCL, pH 7.5, 0.1 M NaCl, and 1 mM PMSF).
- Starter cultures were initiated by inoculating 1.5 ml of FI medium (plus appropriate nutritional supplements and 2% glucose) in sterile 15 ml polythene centrifuge tubes with small portions of colonies, picked by inoculating loop from stock plates. These starter cultures were grown for up to 24 hours at 30°C with shaking (at 120 rpm) and were used as the source of inoculum for the assay cultures. Cells were inoculated into 1.5 ml FI cultures in 15 ml polythene centrifuge tubes to give an initial OD 6 o 0nm of -0.1 (typically a 10-15 ⁇ l inoculum per 1.5 ml culture).
- the lids of the tubes were left one-quarter unscrewed, but held in place by masking tape, to ensure maximum oxygenation for GFP maturation.
- Half of the tubes were treated with 0.005% methyl methanesulphonate (MMS, methanesulphonic acid methyl ester) and half left as unchallenged controls.
- MMS was purchased as a liquid and this stock solution was taken to be 100%. This was diluted to a 0.5% stock in small aliquots as required and then diluted 1:100 for the assay cultures (15 ⁇ l of 0.5% stock per 1.5 ml culture). Assay cultures were incubated at 25°C (30°C when the S65T-GFP was replaced by yEGFP, which is more heat stable) in shaking water bath incubators for 14-16 hours.
- Each of the 1.5 ml cultures was transfe ⁇ ed to a 4-window acrylic cuvette (Sarstedt Ltd, Numbrecht, Germany) and diluted with 1 ml of sterile water before measurement.
- a 4-window acrylic cuvette Session Instachi, Numbrecht, Germany
- the cells were washed twice in sterile water to remove traces of autofluorescent medium and resuspended in 1.5 ml of sterile water. Washed cells were then transferred directly to 4-window acrylic cuvettes containing 1 ml of sterile distilled water. 4-window cuvettes were necessary since measurement of fluorescence emission is performed perpendicular to the path of excitation light.
- Fluorescence measurements were performed with a Perkin-Elmer LS50B Fluorescence Spectrometer (Perkin-Ehner Ltd., Beaconsfield, UK).
- the excitation and emission wavelengths were set to 488 nm and 511 nm respectively, with a slit width of 10 nm, for the S65T GFP derivative- expressing reporter.
- the excitation and emission wavelengths were set to 490 nm and 518 nm respectively (due to the altered fluorescence characteristics of yEGFP), with 5 nm slit widths.
- the OD 60 o nm was recorded for each cuvette.
- the fluorescence values obtained from the fluorometer were then divided by the absorption/scatter readings to give the "brightness value", an arbitrary unit which is independent of sample concentration, though varies with different fluorometers.
- the y-axis on unco ⁇ ected fluorescence scans gives raw data in the form of machine defined fluorescence intensity ('TNT”) units.
- the induction ratio is used to calculate a "signal-to-noise ratio" with respect to the GFP signal.
- All detected signals are not pure GFP signals, but incorporate contaminating background fluorescence signals (auto fluorescence).
- Auto fluorescence significantly varies with the changing growth phase of a cultureand is dependent on the strain background.
- U is the brightness value from untransformed cells i.e. cells not bearing a reporter plasmid (or other plasmid where specified), that have not been exposed to a genotoxin such as MMS, and as such this value represents the uninduced autofluorescence.
- U MMS is the brightness value from untransformed cells that have been exposed to a genotoxin (in this case, MMS). This value represents the genotoxin-induced level of autofluorescence, often larger than that of uninduced autofluorescence.
- T is the brightness value from cells transformed with one of the i 4D5 -GFP reporter plasmids (or other plasmid where specified) that have not been exposed to a genotoxin. This value represents the constitutive level of GFP fluorescence due to continual low-level expression from the RAD54 promoter, and also incorporates the uninduced autofluorescence.
- T MMS is the brightness value obtained from cells transformed with a plasmid that have been exposed to a genotoxin (again, MMS in this case).
- T MMS represents a combination of the GFP signal due to damage-induced up-regulation of expression from the RAD54 promoter, the constitutive GFP signal, and the damage-induced autofluorescence signal.
- the two parameters of interest are the GFP signal due to the damage-induced response of RAD54 and the constitutive GFP response in the absence of damage.
- the constitutive signal is obtained by subtracting the uninduced autofluorescence from T (i.e.
- T — U T MMS - U MMS
- T MMS - U MMS T MMS - U MMS
- Figure 13 shows a schematic diagram of the basic layout of the instrumentation developed with a fluorescence flow cell, through winch the yeast culture or GFP extract is circulated by use of a Gilson Minipuls 3 peristaltic pump (purchased from Anachem Ltd, Luton, UK).
- the 488 nm excitation was provided by an air-cooled 40 mW argon ion laser (LG Laser Graphics GmbH, Dieberg, Germany), reduced to 5 mW by filtering.
- a photomultiplier tube (PMT) was used as the fluorescence detector and a silicon photodiode (SPD) as the scattered light detector for nephelometric measurement of the cell density.
- the flow cell, PMT, SPD, and associated electronics were housed in a light-tight box (in duplicate in Figure 13). The PMT is positioned to one side of the flow cell such that fluorescence is detected at 90° to the path of the excitation light.
- the SPD is situated at the other side of the flow cell from the light source in the path of the excitation light, or to one side of the flow cell in the same way as the PMT, since scattered light can detected in either orientation.
- Data acquisition and manipulation was performed in real time on a personal computer via a 12-bit analogue to digital converter (ADC) and associated software (Pico Technology Ltd., Cambridge, UK).
- ADC analogue to digital converter
- Figure 14 shows the detection of fluorescence polarisation using the flow-through fluorescence detector.
- Polarised laser light polarisation represented by vertical arrows
- fluorescence is detected at 90° on either side of the flow cell with a single polaroid filter between the flow cell and each PMT. Fluorescence from the sample in the flow cell is less polarised than the excitation light (represented by vertical, horizontal, and diagonal a ⁇ ows).
- the polaroid filters to either side of the flow cell are arranged in opposite orientations, such that one filter allows the vertical component of the fluorescence to pass through (parallel orientation, with respect to the plane of polarisation of the incident light), while the other permits transmission of only the horizontal component of the fluorescence (perpendicular orientation, with respect to the plane of polarisation of the incident light).
- I ⁇ is the fluorescence intensity measured polarised parallel to the absorbed plane-polarised radiation, and I is that perpendicular to the absorbed radiation.
- P is a dimensionless parameter and is not dependent on the intensity of the emitted light or on the concentration of the fluorophore.
- FIG. 1 shows the average normal brightness for 3 replicates of FF 18984 cells carrying pWDH445 (darker bars) and the average brightness for 3 replicates of brighter FF18984 cells carrying pWDH445 (lighter bars).
- 'T' transformed cells with no MMS
- 'T MMS' was 9 fold brighter.
- the enhanced brightness in the variant cells actually led to a decrease in the induction ratio from 7.72 to 3.27.
- the ease of detection of the variant cells with enhanced signal output made them attractive for the development of ever-brighter reporters.
- the cells bearing pWDH445 were then tested for resistance to G418, which is conferred by the kanMX module carried on pWDH445.
- Cells of typical brightness were G418 resistant and hence able to grow on G418 (200 ⁇ gml "1 ) containing YPD plates.
- bright variant cells were unable to grow on such medium, despite the previous confirmation of the presence of pWDH445 by growth on uracil-deficient medium. This suggested that mutation or rearrangement of the reporter plasmid had occurred in the variant cells.
- the RAD54-GFV cassette has no gross rearrangements
- Plasmid was prepared from yeast using standards techniques and the DNA pellet was dissolved in 40 ⁇ l of sterile water. 5 ⁇ l of each plasmid preparation was used to transform DH5 ⁇ E. coli cells using standard techniques. E. coli transformed with the plasmids prepared from yeast were selected by growth on LB agar containing 80 ⁇ gml "1 ampicillin. Transformant cultures and plasmid preparations were made as described in the methods and DNA pellets were resuspended in 50 ⁇ l of T ⁇ and RNAase (20 ⁇ gml "1 ).
- coli after amplification was used to re-transform FF 18984 yeast with normal and rearranged pWDH445, using the lithium acetate / PEG / SS-DNA protocol.
- Transformants were selected by their ability to grow on medium deficient in uracil, since this was apparently unaffected by the mutations / rea ⁇ angements in the variants.
- the auxotrophic markers of transformants were checked as before and corresponded to those of the FF18984 background, with the exception of the ability to grow without uracil, confe ⁇ ed by pWDH445. Once more, resistance to G418 was tested by transferal of transformants to YPD plates containing 200 ⁇ gml "1 G418.
- Figure 3 shows photographs taken of re-transformed FF 18984 incubated on YPD plates containing G418 and SD plates lacking uracil.
- the uracil-deficient plates are represented in the right-hand column and as expected demonstrate growth in all cases.
- the G418 plates reveal that cells ca ⁇ ying the control pWDH445 and the normal pWDH445 isolated from yeast are G418 resistant as expected, but cells bearing either of the 2 anomalous plasmids are sensitive to 200 ⁇ gml "1 G418. This suggests that the mutations or rea ⁇ angements are stable in that they were not reverted by the mutational effects sometimes associated with the transformation procedure.
- Re-transformed G418-sensitive yeast still give brighter signal outputs FF18984 cells re-transformed with the altered reporter plasmids were tested for their ability to induce the reporter in response to 0.005% MMS, compared with cells ca ⁇ ying the normal pWDH445. After 15 hours incubation with MMS, fluorescence and scatter measurements were made and brightness values determined.
- Figure 4 depicts the brightness signals from both cells bearing normal pWDH445 (darker bars) and cells conveying the altered plasmids (lighter bars). As previously, the lighter bars for the transformed cells are significantly larger than the corresponding darker bars.
- the modified plasmids gave rise to a 3.3 fold increase in the untreated brightness signal and a 4.6 fold increase in the MMS-induced signal, compared with cells bearing the unchanged pWDH445. Overall these increases had little effect on the induction ratio, producing only a slight decrease from 5.5 to 5.2 in cells bearing modified plasmid. However, the increase in brightness signal with the re-transformants was not as great as for the original variants isolated.
- Xba I digestion revealed size differences in the rearranged plasmids
- Ban ⁇ I and Ase I double digestion employed previously to confirm the presence of the intact HO-RAD54 promoter-j ⁇ EGEP module also revealed that one rearranged plasmid was larger than pWDH445 whilst the other was smaller.
- Xba I digestion was used to confirm the size changes in the rea ⁇ anged plasmids after fractionation of the DNA fragments by agarose gel electrophoresis.
- lanes 1 and 3 represent the fragments created by Xba I digestion of pWDH445 and plasmid from cells of normal brightness, separated on a 1% agarose gel and labelled with ethidium bromide. Both lanes show identical bands in a doublet formation at an approximate size of 6 kb (lane 5 contains 500 bp ladder molecular weight marker DNA, with the largest fragment at 5 kb).
- Lane 4 represents the Xba I fragments for the rea ⁇ anged plasmid that appeared to be smaller in the Bam ⁇ I - Ase I double digestion. The smaller size is also reflected in the replacement of the larger band from the doublet (-6 kb) with a band at -4.4 kb, giving a vector size of -10.4 kb as opposed to the 12 kb of pWDH445.
- Lane 2 shows the Xba I fragments for the other rea ⁇ anged plasmid that appeared to be larger than pWDH445 in the Bani ⁇ I - Ase I digestion.
- the larger band represents the half of the plasmid bearing Amp kanMX3, and yEGFP. This further corroborated the suggestion from the Bam ⁇ I - Ase I digestion, that the HO-RAD54 promoter- EGE module was intact after rearrangement.
- FIG. 7 shows a picture of the ethidium bromide stained 1% agarose gel used to fractionate the Sea I fragments from pWDH445 (lane 2), the larger rearranged plasmid (lane 3), re-isolated pWDH445 (lane 4), and the smaller rearranged plasmid (lane 5).
- Lanes 2 and 4 show the same patterns of bands, with a doublet between 4 and 5 kb (representing the 4.7 and 4.1 kb fragments) and single bands at -1.8 kb and 1.3 kb.
- Neither of the rearranged plasmids (lanes 3 and 5) exhibit the 1.3 kb Sea I band, the production of which requires the Sea I recognition site within kanMX3 to be intact.
- the -1.8 and 4.1 kb bands are present in all 4 of the plasmids digested, implying that no alteration had occurred in the region containing HO, URA3, the 2 micron origin, or Amp r .
- the portion of the vector in which the rearrangements could have occu ⁇ ed was further limited to the region between the unique Ase I recognition site and the Sea I recognition site in Amp ⁇ , including the entire kanMX3 module.
- the Pst I cleavage site within the ka ⁇ MX3 module is lost in the rearranged plasmids Once the region of the plasmid incorporating the rearrangements was established, it was necessary to test restriction enzymes with cleavage sites in this section.
- the Pst I cleavage site occurs 7 times in pWDH445, though only 6 bands were expected to be detectable on an ethidium bromide stained 1% agarose mini-gel after electrophoretic separation of the Pst I fragments. The seventh fragment was too small to be detected under these conditions. Approximate sizes for the 6 bands were 4.2, 2.5, 2.2 or 1.6, 1.4, 1.3, and 0.9 kb.
- the fragment expected to be detected at 2.2 or 1.6 kb should result from cleavage at the sites within HO and URA3, but the size is dependent upon the orientation of the URA3 gene.
- Figure 8 shows a picture of the ethidium bromide stained gel in which the fragments from Pst I digested plasmid were separated.
- pWD ⁇ 445 and re-isolated pWDH445 were loaded into lanes 2 and 4, respectively, whilst the larger and smaller rearranged plasmids were loaded into lanes 3 and 5, respectively.
- All four plasmids exhibit a 1.6 kb band suggesting that the URA3 gene is transcribed in the opposite direction to the HO-RAD54 oxomote ⁇ -yEGFP-kaj ⁇ MX3 cassette.
- the 2 bands involving the Pst I cleavage site in the kanMX3 module are the 2.5 and 0.9 kb fragments.
- the 1.1 kb band results from cleavage by Pvu I in the Amp r and Kan ⁇ genes, suggesting that the Pvu I cleavage site at the 5' end of kanMX3 was intact.
- neither rearranged plasmid shows bands representing the 1.2 and 0.7 kb kanMX3 internal fragments. This suggests that a loss of DNA within the kanMX3 module has occurred.
- the larger rearranged plasmid also showed 2 novel bands at 1 kb and -3.5 kb.
- Sac I does not liberate ⁇ 2.1 kb of kanMX3 from the rearranged plasmids
- Sac I cleavage sites in pWDH445, one in each of the direct repeat sequences flanking Kan T in kanMX3.
- 2 bands representing 2 fragments of -2.1 kb and -10 kb should be produced by Sac I digestion of pWDH445.
- the 2.1 kb fragment is released from kariMX3 whilst the larger fragment represents the remainder of the plasmid.
- the four plasmids were digested with Sac I restriction endonuclease and the resulting fragments were separated electrophoretically in a 1% agarose gel.
- Figure 10 is a picture of the ethidium bromide stained gel with pWDH445 in lane 1, the larger rea ⁇ anged plasmid in lane 2, isolated pWDH445 in lane 3, and the smaller rea ⁇ anged plasmid in lane 4.
- Only lanes 1 and 3 exhibit the expected 2.1 kb fragment released from kanMX3, whilst lanes 2 and 4 both show only a single band representing uncut plasmid. hi conjunction with the other evidence discussed above, this suggests that both rea ⁇ anged plasmids have lost a portion of kanMX3 between and including the Sac I cleavage sites in the direct repeat sequences.
- a 3 ⁇ l aliquot of Sac I-digested pWDH445 was loaded onto a 1% agarose gel and the fragments separated by electrophoresis in order to check the success of the digestion (data not shown). After heat inactivation of the Sac I enzyme by incubation at 65°C for 20 minutes, digested pWDH445 was then re-ligated. Ligations were performed at 3 different concentrations of digested plasmid DNA (undiluted, 1 in 10 fold dilution, and 1 in 100 fold dilution) by incubation with T4 DNA ligase at 16°C overnight. Dilutions of digested DNA were used in order to promote intramolecular ligation events.
- Lane 7 contained a 500 bp ladder molecular weight marker with the largest band at 5 kb, demonstrating that the bands in lanes 1 and 13 were -6 kb.
- Lane 10 contained DNA from the kanamycin resistant colony and reveals the same 6 kb band as the controls in lanes 1 and 13. The remainder of the lanes contained DNA from kanamycin sensitive colonies and all exhibit a band at -6 kb, but also a second band at -4.5 kb. This is the same banding pattern as that seen for Xba I-digestion of the smaller rearranged plasmid (see lane 4, Figure 6).
- the new plasmid is brighter than pWDH445 in yeast FF 18984 cells were transformed with DNA from preparations of the new plasmid using standard methods. Transformants were selected for by incubation on SD plates lacking uracil and the auxotrophic markers were checked. 10 transformants were picked for assessment of brightness in response to 0.005% MMS. Incubation with MMS lasted 15 hours before measurement of fluorescence and scatter. The average brightness values determined for cells bearing the new plasmid (red bars) are compared with those from cells carrying pWDH445 (blue bars) in Figure 12.
- Transformation of FF18984 with pWDH445 generated some transfo ⁇ nants with a 12 fold brighter constitutive signal and a 9 fold brighter MMS-induced signal than typical pWDH445-bearing FF18984 cells. The induction ratio was halved in the brighter cells.
- Double restriction of plasmids isolated from the brighter transformants with BamH I and Ase I produced the same size bands as pWDH445, suggesting that the HO-RAD54 pxomotex-yEGFP module was unchanged. However, one rearranged plasmid was larger than pWDH445 whilst another was smaller.
- Yeast cells (FF18984) re-transformed with rearranged plasmid were sensitive to G418, confirming that the change in brightness was due to the plasmid change, as opposed to a chromosomal mutation.
- Sac I does not release the expected -2.1 kb kanMX3 fragment from the rea ⁇ anged plasmids.
- DISCUSSION Rea ⁇ angements of pWDH445 occurred, probably in a transformation-dependent manner given that transformation is known to be a mutagenic process, generating brighter than normal transformants.
- These bright transformants were found to be sensitive to growth on medium containing 200 ⁇ gml "1 geneticin, despite pWDH445 bearing a kanMX module incorporating the gene encoding the aminoglycoside phosphotransferase that confers resistance to geneticin on yeast cells. This suggested that mutation or loss of the kanMX module had occurred, leaving the cells sensitive to geneticin. It was anticipated that kanMX had probably been lost from the plasmid, since a deletion event would reduce the size of the plasmid, increasing its stability and hence copy number.
- the smaller plasmid was probably generated by recombination between the direct repeats that flank the kan ⁇ gene and promoter sequences within the kanMX module.
- the kanMX fragment released by Sac I digestion is -2.1 kb which is approximately the size of fragment lost by the small plasmid.
- the enhancement of reporter brightness is not related to a reduction in size and hence increase in copy number, since one of the rearranged plasmids was shown to have increased in size.
- the kanMX module is immediately downstream of the HO-RAD54pxomotex-yEGFP cassette with only a direct repeat sequence (465 bp) separating yEGFP from AgTEF.
- pWDH445 bearing kanMX might be such that the plasmid assumes a conformation restricting access of components of the transcriptional complex to the RAD54 promoter.
- loss of kanMX regardless of the mechanism could result in alteration of the conformation, due to the change in size and sequence, leading to greater accessibility of the RAD54 promoter.
- pWDH445 is of the critical size allowing it to assume the restrictive conformation, and hence any change in size, either increase or reduction, alters the conformation sufficiently.
- kanMX An alternative explanation for the enhanced brightness in the absence of kanMX is the greater availability of general transcription factors. Since the aminoglycoside phosphotransferase encoded within kanMX is constitutively expressed, it must have a constant requirement for general transcription factors. Thus, the availability of such factors for transcription from the RAD54 promoter is reduced in the presence of kanMX, and removal of this module eliminates the constraints on the reporter cassette. This is most likely only a "local" deficiency in the volume occupied by the plasmid, and might reflect a higher affinity for the transcription factors in the AgTEF promoter.
- kanMX related interference with reporter expression might occur post- transcriptionally.
- yEGFP and the aminoglycoside phosphotransferase are heterologous proteins since GFP originates from Aequorea victoria and Kan ⁇ from the E. coli transposon Tn903. It is possible that the 2 foreign proteins interact, preventing efficient protein folding and maturation of yEGFP. Loss of kanMX would result in loss of the illegitimate interaction, increasing the proportion of fluorescent yEGFP.
- kanMX The mechanism by which kanMX is lost and reporter output is enhanced is essentially irrelevant to the commercial development of the reporter (as long as the response remains predictable), since a brighter reporter is obviously beneficial.
- the kanMX module was employed as a convenient selectable marker for research purposes, but stringent rules governing the release of genes conferring antibiotic resistance would require its displacement for commercialisation.
- the reporter cassette could be re-cloned into a different plasmid backbone, in order to determine if the effect is specific to pWDH445.
- kanMX with a different gene conferring antibiotic resistance, such as hygromycin B (hph; hphMX), nourseothricin (nat; natMX), and bialaphos (pat; patMX)
- hph hygromycin B
- nourseothricin nourseothricin
- patMX bialaphos
- An intergenic region could be inserted between yEGFP and kanMX on pWDH445 to determine whether the effect is caused by the proximity of the AgTEF promoter of kanMX to the termination codon of yEGFP.
- Assessment of these new vectors would be by constitutive and induced brightness values when borne by the FF 18984 strain.
- Bright transfo ⁇ nants have been isolated from other yeast strains though plasmid DNA has not been isolated and assessed in the same way as from bright FF18984 cells. However, it is anticipated that similar rea ⁇ angements would be detected in plasmid isolated from alternative strains, which would discount a strain-specific effect.
- GSA GreenScreen ® assay
- the GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico Structure Activity Relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.
- SAR Structure Activity Relationship
- RAD54-GFV yeast-based genotoxicity test system
- Induction of the RAD54 promoter results in the production of the extremely stable Green Fluorescent Protein (GFP), which is fluorescent in the green spectrum when illuminated by blue light. It appears that any agent able to cause mutation in yeast will lead to RAD54 induction.
- GFP Green Fluorescent Protein
- the specificity to DNA damage has been confi ⁇ ned by studying the global transcriptional response to DNA-damaging agents using DNA microarrays. It was found that RAD54 did not respond to other non-genotoxic stresses, such as heat shock, oxidative stress, reductive stress, osmotic shock or amino acid starvation.
- RAD 54 encodes a structural element of the homologous recombinational (HR) repair pathway, but responds to a broad spectrum of genotoxins, suggesting that the yeast DNA damage sensing pathways activate the HR pathway as a default for failure of the other repair pathways. This is supported by several lines of genetic evidence. For example the loss of the non-homologous end-joining (NHEJ) pathway in yeast is undetectable unless the RAD52 (HR) pathway is also ablated. Furthermore, it has been demonstrated that induction of RAD54 by methyl methanesulfonate (MMS) is insensitive to mutations in the 'classical' DNA-damage sensing pathway controlled by the RAD9 and DDC1 checkpoints.
- MMS methyl methanesulfonate
- yeast strains, plasmids and growth media (FI) used in tins study have been described previously (Walmsley et al, 1997) and the use of Green Fluorescent Protein (GFP) as a reporter for the DNA damage-induced gene RAD54 from Saccharomyces cerevisiae, has also been described (Billinton, et al., 1998).
- the Saccharomyces cerevisiae strain FF18984 MATa, leu2-3,112 ura3-52 lys2-l his7-l was obtained from Francis Fabre (French Atomic Energy Commission (CEA), Fontenay-aux-Roses, France).
- the reporter strain is FF18984 containing a replicative plasmid (pGenOOl shown in Figure 15) containing the entire upstream non-coding DNA sequence of the RAD54 gene fused to the yeast-enhanced Aequorea victoria GFP gene.
- the control strain is FF 18984 containing an identical plasmid except that 2 base pairs have been removed at the start of the GFP gene, such that no GFP is made.
- Microplate preparation Assays were carried out in 96 well, black, clear-bottomed microplates (Matrix ScreenMates, Cat. No. 4929, Apogent Discoveries, USA). A number of alternative microplates were assessed, though the variable background absorbance and fluorescence both within and between plates from individual manufacturers were generally unacceptable, leading to the conclusion that only Matrix or Corning (BV, Netherlands: Cat. No. 3651) plates were appropriate for this assay.
- the assays were performed using a liquid handling robot (MicroLabS single probe, Hamilton GB Ltd., Birmingham. UK) in a protocol designed to set up 4 compounds per test on a single 96 well microplate in 30 minutes.
- a microplate version of the assay has been previously reported (Afanassiev et al, 2000), but different microplate layouts and controls have been used for this study, so more detail is presented here.
- the following standard protocol was followed.
- a 1 mM stock of the test chemical was prepared in 2% v/v aqueous DMSO, and used to make 2 identical dilution series across the microplate and a 'control' (see below).
- 150 microlitres of the test chemical solution were put into 2 microplate wells.
- Each sample was serially diluted by transferring 75 microlitres into 75 microlitres of 2% DMSO, mixing, and then taking 75 microlitres out and into the next well. This produced 9 serial dilutions of 75 microlitres each.
- the cytotoxicity threshold is set at 80 % of the cell density reached by the untreated control cells. This is greater than 3 times the standard deviation of the background.
- a positive cytotoxicity result (+) is concluded if 1 or 2 compound dilutions produce a final cell density lower than the 80% threshold.
- a strong positive cytotoxicity result positive (++) is concluded when either (i) three or more compound dilutions produce a final cell density lower than the 80% threshold or (ii) at least one compound dilution produces a final cell density lower than a 50% threshold.
- a negative result (-) is concluded when no compound dilutions produce a final cell density lower than the 80% threshold.
- the lowest effective concentration (LEC) is the lowest test compound concentration that produces a final cell density below the 80% threshold.
- the compound absorbance control allows a warning to be generated if a test compound is significantly absorbing. If the ratio of the absorbance of the compound control well to a well filled with diluent alone is > 2, there is a risk of interference with interpretation.
- the cytotoxicity controls indicate that the yeast is behaving normally.
- the 'high' methanol standard should reduce the final cell density to below the 80% threshold, and should be a lower value than the 'low' standard.
- the genotoxic threshold is set at a relative GFP induction of 1.3 (i.e. a 30% increase). This is greater than 3 times the standard deviation of the background.
- a positive genotoxicity result (+) is concluded if 1 or 2 compound dilutions produce a relative GFP induction greater than the 1.3 threshold.
- a strong positive genotoxicity result (++) is concluded if either (i) three or more compound dilutions produce a relative GFP induction greater than the 1.3 threshold or (ii) at least one compound dilution produces a relative GFP induction greater than a 1.6 threshold.
- a negative genotoxicity result (-) is concluded where no compound dilutions produce a relative GFP induction greater than the 1.3 threshold.
- the LEC is the lowest test compound concentration that produces a relative GFP induction greater than the 1.3 threshold.
- the genotoxic controls demonstrate that the strains are responding normally to DNA damage.
- the 'high' MMS standard must produce a fluorescence induction > 2, and be a greater value than the 'low' MMS standard.
- Anomalous brightness data is generated when the toxicity leads to a final cell density less than 30% that of the blank. Genotoxicity data is not calculated above this toxicity threshold. Compounds that tested negative for genotoxicity, were re-tested up to lOmM, or to the limit of solubility or cytotoxicity.
- the compound fluorescence control allows a warning to be generated when a compound is highly auto-fluorescent. If the ratio of the fluorescence of the compound control well to a diluent filled well is >5, there is a risk of interference with interpretation. In these cases (four in this study), fluorescence polarisation can be used to distinguish GFP from other sources of fluorescence (Knight et al, 2000, 2002). Both the Tecan and BMG instruments have this facility. Occasionally, compounds though not fluorescent themselves, induce cellular auto-fluorescence. This is apparent from rising brightness in the control (GenCOl) strain in the absence of fluorescence from the chemical-only control. The routine subtraction of GenCOl from GenTOl data removes this interference from the data.
- IARC International Agency for Research on Cancer: http://www.iarc.fr
- NIOHS National Institute for Occupational Health and Safety: http://www.cdc.gov/niosh/homepage.html
- USEPA Environmental Protection Agency: http ://www. epa. gov/iris/search.htm
- Table 12 Suggested maximum tolerable solvent concentrations.
- Table 12 lists chemicals/solvents that are often used with test samples, to promote dissolution etc. All of these compounds are toxic at higher concentrations due to osmotic effects and/or effects on membranes, and interfere with data interpretation. If these chemicals are used to dissolve compound stock solutions, they should also be included in the diluent used to make the compound dilution series.
- the routine use of DMSO in this study revealed its predictable interference with certain compounds. For example, cisplatin was only positive when retested using water in place of 2% DMSO as the diluent. Interference of DMSO, a free radical scavenger, in the recombinogenic properties of several oxidising agents has previously been reported.
- the test with GSA can have a positive outcome, for which there may be either a positive result ('a') or a negative result ('b') from another test (for example MLA).
- the total number of positives for GSA is thus 'a + b'.
- the test with GSA can have a negative outcome, for which there might be either a positive ('c') or a negative ('d') result form the second test.
- the total number of negatives for GSA is thus 'c + d'. It follows that the total number of positive results from the second test is 'a + c' and the total number of negative results from the second test in 'b + d'. similarly the total number of compounds for which there is data for both tests 'n' is 'a + b + c + d'. The following terms were calculated from these data:
- Sensitivity a/(a+c); specificity, d/(b-l-d); predictive value, a/(a+b); prevalence, (a+c)/n
- Figure 21 contains the subset of data for which there are Ames data, and highlights the distinctively different endpoints for GSA and bacterial genotoxicity results.
- the photomutagen psoralen was a GSA positive as the test was only exposed to normal laboratory lighting.
- the reporter also responds to a variety of genotoxins that may not act directly on DNA. These include compounds that target topoisomerases (etoposide, ellipticine) as well as anti-mitotic, spindle targeting compounds (aneugens) such as colchicine and econazole.
- aneugens such as colchicine and econazole.
- the spectrum of metabolic enzymes expressed by yeast is less complex than that of mammalian cells.
- CYP cytochrome P450
- the CYP type enzymes ECG5 / CYP61; DIT2 / CYP 56; P450 14 -DM / ERG11 / CYP 51; the P450 oxido-reductase NCP1 / CPR1 and glutathione-S-transferases GTT1 and GTT2.
- yeast contrasts to the enhanced activities in S9 required for the activation of most promutagens, and explains the most obvious group of compounds not detected by GSA: primary aromatic amines and aromatic amides. 9 such compounds were tested. Two were positive in GSA: 2-amino-4-nitrophenol and 1-naphthylamine. The others were negative in GSA and positive in other in vitro tests: 2-acetamidofluorene, 9-aminoacridine, 2-aminoanthracene, 4,4-oxydianiline, o-anisidine, aniline and 4-aminophenol. The last two of these are negative with and without S9 in Ames.
- the GSA has a simple and fixed microplate protocol with clear decision- making thresholds. Together these properties were conceived to give a high throughput capability and to minimise the generation of conflicting test data. These are important requirements of any useful screen.
- the compounds tested in this screen provide sufficient data to reveal that a positive result with GSA provides a relevant warning of positive results in the regulatory battery of tests, and in the present form provides complementary data to bacterial tests - each providing significantly non- overlapping endpoints. It will be interesting to see how the definition of the endpoint develops as more data become available.
- Sikorski RS, Hieter P A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27, 1989.. Walmsley, R.M., Billinton, N. and Heyer W.-D. (1997). Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae. Yeast, 13, 1535-1545.
- Table 13 A comparison of LEC data from GSA, umuC and lux assays.
- MNNG N-Methyl-N -nitro-N-nitrosoguanidine
- This example provides a prefened protocol for ca ⁇ ying out the method of the invention using a plate assay procedure.
- GenCOl control strain
- GenTOl the test strain
- Each starter culture is prepared as follows : Into a sterile 250 ml conical flask place - 10 ml sterile water 10 ml 2X FI media (See Appendix 1) 1 x 250 ⁇ l colour coded aliquot of frozen cells Seal flask with foam bung (taped to the flask) before incubating.
- test compound PREPARING THE TEST COMPOUNDS. Standard solutions of the test compounds should be freshly prepared shortly before each test run. Unless a specific test concentration is required, the recommended top concentration of a test compound is 1 mM in 4 % DMSO v/v in water. It is necessary to ensure that the final concentration of test compound is made up in the same diluent as that supplied to the robot for serial dilutions. The diluent is 4 % DMSO v/v in water by default but can be substituted for water alone.
- test compound solution 1 ml is required per 'test run' and there is sufficient capacity on a 96-well plate for 4 test runs. Therefore for each plate prepare 2 tubes each containing 1 ml of the respective test compound, label them, cover with black caps and leave in a fume cupboard until required. For reference record the details of the test compound, its concentration, dilution calculation and the diluent used.
- MMS and Methanol prepared from 100% stock solution with a diluent of 4 % DMSO / water (v/v).
- iii Load the GREEN RACK with the appropriate tubes containing yeast cultures, samples, standards, 90 % ethanol, plate blank and an addition one for waste volumes of the test compounds according to the loading sequence (see figure 21).
- iv. Place the appropriate diluent solutions in position to the right hand side of the deck with tube lines connected to the conesponding MVP selector positions. (Position 1 by default).
- v. Place the 'clean wash' tube in a container of sterilised water (approx. 300 ml per plate).
- vii. Remove all tube caps and place in a suitable disinfectant / compound neutralising solution (i.e.
- PLATE READING AND DATA ANALYSIS For plate reading and analysis refer to the protocol appropriate for the plate reader used. i.e. Tecan Ultra or BMG Polar Star.
- EXAMPLE 4 ha addition to the RAD54 vectors described in the previous Examples, the inventors constructed an FF18984 reporter strain containing a replicative plasmid harbouring an RNR regulatory element.
- the vectors pGenRNR2 or pGenRNR3 are shown in Figures 24 and 25, respectively, and contain the upstream non-coding DNA sequence of the RNR2 gene or RNR3 gene fused to the yeast-enhanced Aequorea victoria GFP gene.
- Figure 28 illustrates results obtained in a yeast test strain transformed with pGenRNR2. Brightness/fluorescence was induced by the genotoxic compound MMS. The inventors believe this compound activates the RNR2 regulatory element which in turn results in the expression of GFP and thereby produces a signal measurable according to the method of the invention
- Figure 29 illustrates data similar to that shown in Figure 28, except that the yeast strain was transformed with pGenRNR3.
- FIGS 30 to 32 show graphs of the response of RNR3-GFP reporter to MMS, 1,2- dimethylhydrazine (2HC1), and EMS, respectively. In each case, it will be seen that the fluorescence induction by the reporter increases with increasing concentration of test compound.
- the inventors also constructed a vector according to the first aspect, using as backbone, the vector pRS316, which is a centromeric plasmid.
- pRS316 is URA3 selected.
- Figure 39 shows empty pRS316 vector compared with pRS316 containing the RAD54-GFP reporter cassette plus the kanMX module, compared with pRS316 containing the RAD54-GFP cassette but not kanMX. It will be appreciated that pRS316 containing the RAD54-GFP cassette but not kanMX gives a brighter reporter signal.
- the brightness values are lower than pWDH445, since pRS316 is a cenfromeric plasmid i.e. at a low copy number.
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AFANASSIEV V ET AL: "Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances" MUTATION RESEARCH, AMSTERDAM, NL, vol. 464, no. 2, 24 January 2000 (2000-01-24), pages 297-308, XP002302121 ISSN: 0027-5107 * |
BILLINTON N ET AL: "DEVELOPMENT OF A GREEN FLUORESCENT PROTEIN REPORTER FOR A YEAST GENOTOXICITY BIOSENSOR" BIOSENSORS & BIOELECTRONICS, ELSEVIER SCIENCE PUBLISHERS, BARKING, GB, vol. 13, no. 7/8, 1998, pages 831-838, XP001145519 ISSN: 0956-5663 * |
WACH A ET AL: "New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae" YEAST, CHICHESTER, SUSSEX, GB, vol. 10, no. 13, November 1994 (1994-11), pages 1793-1808, XP002108452 ISSN: 0749-503X * |
WALMSLEY R M ET AL: "GREEN FLUORESCENT PROTEIN AS A REPORTER FOR THE DNA DAMAGE-INDUCED GENE RAD54 IN SACCHAROMYCES CEREVISIAE" YEAST, CHICHESTER, SUSSEX, GB, vol. 13, December 1997 (1997-12), pages 1535-1545, XP002073536 ISSN: 0749-503X * |
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